WO2008112980A2 - Procédé et système pour l'assemblage de macromolécules et de nanostructures - Google Patents

Procédé et système pour l'assemblage de macromolécules et de nanostructures Download PDF

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WO2008112980A2
WO2008112980A2 PCT/US2008/057013 US2008057013W WO2008112980A2 WO 2008112980 A2 WO2008112980 A2 WO 2008112980A2 US 2008057013 W US2008057013 W US 2008057013W WO 2008112980 A2 WO2008112980 A2 WO 2008112980A2
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template
dna
single strand
amino acid
ssdna
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PCT/US2008/057013
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WO2008112980A3 (fr
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Vincent Suzara
Paul Bentley
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Incitor, Llc
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Priority claimed from US11/936,045 external-priority patent/US20090118140A1/en
Application filed by Incitor, Llc filed Critical Incitor, Llc
Publication of WO2008112980A2 publication Critical patent/WO2008112980A2/fr
Publication of WO2008112980A3 publication Critical patent/WO2008112980A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic

Definitions

  • the present invention relates generally to synthetic methods and systems for the in-vitro template-mediated synthesis of macromolecules, e.g., polypeptides, enzymes, nanostructures, and the like.
  • macromolecules e.g., polypeptides, enzymes, nanostructures, and the like.
  • These template-based systems can be used to synthesize new and improved enzymes, including, for example, cellulases, which are important enzymes in the manufacture of ethanol from plant biomass.
  • Plant biomass e.g., agricultural and forestry products, associated by-products and waste, municipal solid waste, and industrial waste, is the most abundant source of carbohydrate in the world due to cellulose-rich cell walls of all higher plants.
  • Cellulose can be converted to sugars, which are ultimately fermented to ethanol by well-known methods.
  • a major limitation in ethanol production is the severe intolerance of cellulose-degrading enzymes to high-acid and high-temperature conditions typical of ethanol production processes as befitting their nature as biodegradable molecules.
  • template-based systems known in the art relates, in part, to their inability to precisely, accurately, and efficiently manipulate, on a molecular scale, the molecular building blocks comprising the macromolecules and nanostructures of interest such that new and useful macromolecular structures (e.g., novel or improved enzymes) and nanostructures (e.g., one-, two-, and three-dimensional arrays for use in micromechanical, microelectronic, bioelectronic and bio-sensing applications) can be developed.
  • macromolecular structures e.g., novel or improved enzymes
  • nanostructures e.g., one-, two-, and three-dimensional arrays for use in micromechanical, microelectronic, bioelectronic and bio-sensing applications
  • the present invention relates generally to methods and systems for the template-mediated synthesis of macromolecules and nanostructures, and to the macromolecules (e.g., peptides, proteins and enzymes) and the nanostructures synthesized by the herewith methods and systems.
  • the present invention further relates to using the macromolecules and nanostructures prepared by the methods and devices of the invention for useful processes, for example, cellulose degradation and other enzymatic processes.
  • the present invention is directed to a template-based system for assembling a macromolecular structure comprising a surface comprising a plurality of single strand DNA molecules which are substantially parallel, substantially inline each from one end, and substantially equally spaced apart, wherein each DNA molecule has a distinguishable length and a known sequence.
  • the macromolecular structure assembled by the system can be a polypeptide, e.g., an enzyme, such as, cellulase.
  • the macromolecular structure assembled by the system can also be a nanostructure.
  • the polypeptides of the invention can comprise a secondary skeleton, including (a) thiol-maleimide linkages at one or more residues, (b) thiol to gold linkages at one or more residues) and/or (c) cyclized thiol linkages between two or more residues.
  • the surface in one aspect, can be gold.
  • the single strand DNA molecules can comprise alpha-Sulfur single strand DNA molecules or alpha-Sulfur oligonucleotides bound to the gold surface.
  • the present invention is directed to a method for preparing a template-based system for assembling a macromolecular structure, comprising the steps of: (a) providing a substrate having a surface and a doormat region, (b) providing a plurality of single strand DNA molecules, each having a distinguishable length and a known sequence and each having a bead bound to one end and each bound at the other end to the doormat region, and (c) stretching the plurality of single strand DNA molecules so that they are substantially parallel, substantially inline each from the doormat region end, and substantially equally spaced apart on the surface.
  • the single strand DNA molecules can be stretched by applying an electrical force acting on the negatively charged phosphate backbone of the DNA, applying a magnetic force acting on the backbone and/or a magnetic bead, and/or applying a centrifugal force acting on the bead.
  • the single strand DNA molecules can comprise alpha-Sulfur single strand DNA molecules that are bound to a gold surface to provide the template-based system.
  • alpha-Sulfur oligonucleotides can be hybridized to their complement nucleotides of the single strand DNA molecules, the hybridized alpha-Sulfur oligonucleotides can be bound to a gold surface, and the bound alpha-Sulfur oligonucleotides can be released from the single strand DNA molecules to provide the template-based system.
  • the method can further comprise hybridizing oligonucleotides to their complement nucleotides of the alpha-Sulfur single strand DNA molecules of the template-based system, encapsulating the hybridized oligonucleotides with an elastomer, and releasing the hydridized nucleotides from the alpha-Sulfur oligonucleotides and the gold surface to provide a platform DNA system.
  • the present invention is directed to a method for assembling a macromolecular structure comprising the steps of: (a) preparing a surface comprising a plurality of single strand DNA molecules which are substantially parallel, substantially inline each from one end, and substantially equally spaced apart, wherein each DNA molecule has a distinguishable length and a known sequence, (b) sequentially hybridizing nucleotide-coupled amino acid chimaeras to complementary nucleotides of the single strand DNA molecules, (c) forming covalent bonds between each adjacent amino acid to form the macromolecular structure, and (d) disassociating the macromolecular structure from the coupled nucleotides.
  • the macromolecular structure can be a polypeptide, e.g., an enzyme, such as, cellulase.
  • the method can further comprise the step of attaching a secondary skeleton to the polypeptide via sulfur linkages at one or more amino acid residues.
  • the secondary skeleton can comprise one or more linkages selected from the group consisting of: (a) thiol-maleimide linkages at one or more residues, (b) thiol to gold linkages at one or more residues, (c) cyclized thiol linkages between two or more residues, and combinations thereof.
  • FIG. 1 shows a top-view schematic illustration of a master DNA system comprising a plurality of parallel, straightened, and stretched single strands of ssDNA in a doormat configuration.
  • FIG. 2A shows an individual stand of single strand DNA functionalized at the 5' end with thiol and at the 3' end with biotin that is complexed to a streptavidin-functionalized magnetic bead.
  • FIG. 2B shows two exemplary methods to functionalize the single strand DNA with 5'-thiol and 3'-biotin.
  • FIG. 3 shows a perspective-view schematic illustration of a gold transmission electron microscope (TEM) grid that has been functionalized to provide a "doormat region" for the single strands of ssDNA to bind to via a MUAM/SSMCC linker to a thiol group.
  • FIG. 4 shows the asymmetric localization of a single strand DNA molecule with its thiol end attached to a gold surface via a MUAM/SSMCC linker.
  • TEM gold transmission electron microscope
  • FIG. 5 shows a coiled-globule ssDNA, end-labeled with biotin and thiol and attached to a streptavidin- coated magnetic bead and a gold surface via linker molecules (top), and the same ssDNA molecule in a partially stretched configuration (bottom).
  • FIGS. 6A to 6E show a schematic illustration of a method to prepare a template DNA array from a master DNA array.
  • FIG. 7 shows the reaction of deoxyadenosine monophosphate with arginine via a cystamine linkage to provide a chimaera of the amino acid linked to the nucleotide.
  • FIG. 8A shows a schematic illustration of the basic concept of bonding of a chimaera, comprising an amino acid and a nucleotide, to a template DNA strand by nucleotide pairing.
  • FIG. 8B shows a schematic illustration of a fully realized polypeptide sequence as attached to the template DNA strand.
  • FIGS. 9A and FIG. 9B show a method of synthesizing a polypeptide using a template DNA system, wherein the resultant polypeptide is covalently linked to its nucleotide chimaeric partners and thus can be complexed to either a secondary skeleton via a scaffold with DNA binding capacity or to a template with similar nucleotide sequence.
  • FIGS. 1OA, FIG. 1OB, and FIG. 1OC show a method of synthesizing a polypeptide using a template DNA system, wherein the resultant polypeptide is covalently linked to its template DNA strand through cystamine linkages, which can be utilized as shown to complex to, or self-polymerize to resultantly create, a secondary backbone for scaffolding purposes.
  • FIG. 1 is a top-view schematic illustration of a master DNA system 10 of the present invention.
  • the master system 10 can be prepared by stretching single strands of DNA to prepare a surface comprising a plurality of single strand DNA (ssDNA) molecules 12 that are substantially inline, substantially adjacent, and substantially parallel.
  • the DNA strands 12 can be of the same length or of different length (as shown).
  • One end of each strand 12 is bound to a surface or "doormat region" 14.
  • the other end is bound to a bead 16 that facilitates the stretching of the ssDNA molecules 12.
  • the master DNA system 10 can be formed on the surface of a plastic film or other substrate 18. Described below is an exemplary method that can be used to fabricate the master DNA system 10.
  • FIG. 2A is a schematic illustration of a ssDNA 12 of a specific length that is modified on one end with thiol (-SH) and on the other end with the molecule biotin (-B).
  • ssDNA can be 100 to 10,000 base pairs long and can be 5'-thiolated and 3'-biotinylated (as shown).
  • the ssDNA can be generated by either (1 ) end-labeling of restriction-endonuclease-digested dsDNA with hybridized dsDNA oligonucleotides, resulting in biotinylation and thiolation of one strand (plus +), or (2) polymerase chain reaction (PCR) with a 5'(-SH)-labeled primer, followed by melting of the PCR product into ssDNA, and T4 RNA ligation of the thiolated strand with a 3'(Biotin)-labeled oligonucleotide.
  • PCR polymerase chain reaction
  • FIG. 2B shows two exemplary methods for the functionalization of single strand DNA with 5'- thiol and 3'-biotin.
  • the methods are directed to functionalization of the upper, or plus (+) strand.
  • double strand DNA linkers are ligated specifically to the ends of (1-1 ) in a reaction catalyzed by DNA Ligase, such that the positive (+) strand receives a 5'-thiol and 3'-biotin function, respectively (1-2).
  • the double strand DNA is then converted to single strand DNA, by standard methods such as heat and extremes of pH and/or salt concentration, into the desired product molecule (1-3).
  • the starting material (1-1 ) can also be used as a template for polymerase chain reaction (PCR) utilizing a 5'-functionalized thiol primer and a reverse primer having no unique function (2-1 ).
  • the intermediate molecule (2-2) can be 3'-biotin-functionalized by single strand ligation of a primer having that function as shown (2-3), in a reaction catalyzed by T4 RNA Ligase.
  • Thiol and biotin groups can be functionalized on either the 5' or 3' ends of the product molecule by variations on the above methods.
  • the product molecules shown (1-3 and 2-3) need not necessarily be similar in the sequence of the primers used for final functionalization and/or intermediate processing (as shown in 1-2, 2-1 and 2-2).
  • the present invention is not limited to any particular method of preparing ssDNA, or any method of biotinylation, or thiolation.
  • Other labels are within the scope of the present invention, so long as the particular labels that are used enable one of ordinary skill in the art to localize the ssDNA to a "doormat" configuration (Ae., substantially inline, substantially adjacent, and substantially parallel ssDNA molecules joined each at one end to a surface).
  • alternative labels include a dioxigenin (DIG) ligand binding to an anti-DIG antibody (Smith et al., Science 258, 5085 (1992)) or an amine ligand binding to a primary aldehyde-containing receptor (Fixe et al., Nucleic Acids Research, page 32 (2004)).
  • DIG dioxigenin
  • the former can be used for non-covalent bond-based linking of ssDNA to a bead, whereas the latter can be used for covalent bonding between the ssDNA and the bead.
  • Magnetic beads 16 covered with the molecule streptavidin (-SA) can be complexed with the biotinylated ssDNA molecules to form a ssDNA magnetic bead molecule.
  • the magnetic beads 16 can have a mean diameter of 50 nm.
  • the present invention is not limited to 50 nm-sized magnetic beads and can utilize any usefully-sized beads so long as they allow the ssDNA molecules 12 to be manipulated by magnetic, electrical, gravitational, optical, and/or centrifugal fields, referred to herein as "translocational forces," to facilitate their localization to the "doormat" configuration.
  • the beads are preferably of a usable size, mass, and susceptibility to be translocated by such fields, and also able to bind biotin.
  • the beads can comprise a non-magnetic material, wherein the ssDNA can be pulled simply by the greater mass of the bead, or an optically-sensitive glass or plastic, where the ssDNA can be pulled by virtue of coherent light sources.
  • FIG. 3 is a perspective-view schematic illustration of the preparation of a gold transmission electron microscope (TEM) grid that can be used to provide a gold "doormat region" 14 for the ssDNA 12 to bind to (for ease of illustration, only one strand 12 of a master system 10 is shown in FIG. 3).
  • the gold TEM grid 22 was in a square mesh pattern, like a net, and has a compass marker in its middle to indicate direction and orientation. Only a portion of a single square mesh is shown in FIG. 3.
  • the doormat region 14 can be prepared by forming a protective upper layer 24 on which ssDNA can be blocked from binding to the grid.
  • the thin layer 24 covers most of the grid walls, leaving a thin, narrow edge (the "doormat region" 14) at the base of the grid 22 still exposed and capable of binding to thiolated DNA.
  • the height of the doormat region is less than about 100 nm.
  • a gold TEM grid (e.g., PELCO ® 400 mesh Au TEM, Redding, California) is thoroughly cleaned with hot water, chloroform, and ethanol, and vacuum dried.
  • the top of the grid is covered with a thick layer of liquid Butvar, the solvent for which is chloroform.
  • the layer thickness can be about 20 microns (Ae., the grid thickness).
  • the inner surfaces of the gold TEM grid can be modified with intermediate (linker) molecules to bind the thiolated ends of ssDNA. This can be done in a way such that the ssDNA preferentially bind substantially to the bottom, doormat region, of the grid, and not substantially to the tops or sides.
  • FIG. 4 shows a schematic illustration of the functionalization of the doormat region 14 of the gold TEM grids with MUAM and SSMCC linker molecules.
  • the heterobifunctional linker SSMCC is used to attach 5'-thiol modified oligonucleotide sequences to reactive pads of MUAM.
  • the doormat can be prepared by coating the bottom of grid with MUAM in ethanol. The ethanol very slightly dissolves some of the Butvar plastic on the inner walls of the bottom of the grid, thereby creating the "doormat" by capillary action.
  • the thick Butvar layer can then be peeled off with a forceps and the grid examined with an optical microscope to ensure that no plastic remains.
  • the SSMCC linker contains a N-hydroxy-sulfo-succinimide (NHSS) ester functionality (reactive towards amines) and a maleimide functionality (reactive towards thiols).
  • NHSS N-hydroxy-sulfo-succinimide
  • maleimide functionality reactive towards thiols
  • Dry Butvar is used so that the MUAM- SSMCC linker and MUOL blocker are not dissolved by a chloroform solvent.
  • Any plastic film that is TEM transparent, able to be surface-modified with chemicals that enable DNA stretching, and able to withstand translocation forces can be used for this step.
  • ethylene vinyl acetate (EVA) is another suitable plastic film.
  • ssDNA-magnetic bead molecules can be added to the TEM grids coated with the plastic film.
  • the ssDNA can be (5' or 3')-thiol-terminated and (3' or 5')-biotin-terminated and pre-linked to SA- coated beads.
  • the linker-functionalized grids can be spotted with 5'-thiol-modified ssDNA that reacts with the maleimide groups, forming a covalent bond to the surface monolayer of linker molecules to provide the bound DNA strands.
  • the solvation liquid environment
  • MUAM-SSMCC thiolated end of the ssDNA bind to the linker molecules
  • the solvation conditions can be changed by neutralizing the reduction potential in the buffer in which the ssDNA-magnetic bead molecules are solvated (10 mM DTT in 1X T4 RNA Ligase Buffer) with a redox equivalent amount of H 2 O 2 and then changing the buffer to 10 mM phosphate / 20 mM EDTA / 100 mM NaCI.
  • This resolvation preserves the biotin-SA bonds, yet promotes thiol binding to the maleimide groups on the linkers.
  • Other solvation conditions can be used to bind the ssDNA.
  • the ssDNA 12 bind on the inner sides of the grid, and close to the bottom near the transparent plastic film, i.e., in a "doormat" configuration.
  • the plastic film 18 can be coated, for example with poly-L-lysine (PLL) to give it a slightly positive charge so that the ssDNA will bind tightly to the Butvar with the anionic phosphate groups side- down and the cationic bases side-up after stretching.
  • the concentration of the PLL can be selected to give the Butvar about one positive charge for each negative charge contributed by the doormated DNA. For example, assuming 100,000 strands of 6000 nt ssDNA, the Butvar can be coated with 100 ppm poly-L-lysine (PLL) in water for 2 hours and in 100% relative humidity, followed by a rinsing with water. The washing leaves a mono-molecular coating of PLL on the Butvar.
  • a magnetic field (e.g., by using a hand-magnet) can be applied towards one side of the square mesh pattern (the "left" side) of the grid, and also downwards at a 45° degree angle.
  • This magnetic field facilitates the ssDNA 12 (each bound to a magnetic bead 16) to bind only the bottoms of the inner sides of the TEM grids, and also only to the left side in FIG. 3, i.e. the "doormat” configuration. Therefore, the ssDNA molecules bind at one end “on the bottom of a door” rather than “running up the wall” to the ceiling or "across the floor” on the Butvar. Binding of the DNA only to the doormat region enables quality assurance determination of the DNA sequence, as will be described later.
  • the solvation can be changed to one wherein the ssDNA molecules tend to be in less of a tangled/coil-like configuration, e.g., by changing to 10 mM phosphate buffer, pH 6.8. This resolvation preserves both the biotin-SA bonds and the thiol-maleimide bonds.
  • FIG. 5 is a schematic illustration of a coiled-globule ssDNA 12', end-labeled with biotin and mounted to the doormat region 14 of the gold TEM grid via the linker molecules, and a partially extended ssDNA 12" pulled in the (+x) direction as a result of translocation forces.
  • the translocation force is from right to left in this illustration.
  • the initial elongation (stretching) of the "doormated" ssDNA away from the TEM doormat region in the (+x) direction can be due to any combination of electrical forces acting on the negatively charged phosphate backbone, magnetic forces acting on the backbone and/or the magnetic bead, or inertial (e.g., centrifugal, centripetal, or gravitational) forces acting on the bead. See http://xpcs.physics.yale.edu/boulder1/node11.html and http;//xxx.lanl.gov/PS_cache/cond- mat/pdf/o111/0111170.pdf.
  • the bound DNA 12 can be initially stretched by applying an electrical force acting on the negatively charged phosphate backbone of the DNA.
  • the grids with DNA can be placed in a self-made electrical cell (e.g., prepared as a glass microscope slide with a 1.2 cm square trough, silver electrodes 1 cm apart attached to two AAA batteries and measured with an electrometer) and a small electric field (e.g., approximately 7 Volts and 1 mAmp) facilitating an initial stretch of the DNA molecules. After about 30 minutes, the electric field can be turned off.
  • the bound DNA 12 can then be stretched by a magnetic force acting on the backbone and/or magnetic bead. Therefore, a second magnetic field (e.g., applied with a hand-held magnet) can be directed from left-to-right (Ae., doormat region on the left, with ssDNA being stretched to the right as shown in FIG. 3) and slightly upwards (Ae., in the +z direction, going away from the ssDNA on the surface), in order to do a longer term stretching of the doormated ssDNA.
  • the slight upwards directional vector of the magnetic field helps pull the ssDNA away from the plastic surface, which otherwise would inhibit stretching because the positively-charged surface would strongly adhere to the negatively-charged DNA.
  • This "left-to-right and upwards" magnetic field can be left on for about 8-16 hours to pull the DNA to nearly their fully extended lengths.
  • the solvation environment of the DNA can be changed to that of a less polar nature (e.g., 20% [vol] glycerol in 10 mM phosphate buffer, pH 6.8), which helps keep the ssDNA strands from assuming entangled conformations, as well as decreasing intra-strand hydrogen bonds (Ae., "stickiness").
  • an upper solvent coating of hydrophobic liquid such as mixed hexanes, can be added to the ssDNA that is being stretched to prevent evaporation of the lower aqueous solvent during the hours of stretching.
  • the bound DNA 12 can finally be stretched by a centrifugal force acting on the bead.
  • the grids can be placed in a centrifuge for the final straightening using the weight of the magnetic beads as an "anchor.”
  • the orientation of the grids can be verified by looking at the compass marker under light microscopy, and the grids can be placed into centrifuge mounts that hold the thin, gold grids in place securely without warping.
  • the hydrophobic layer e.g., a previously added layer of mixed hexanes
  • can be sheared-off (ablated) leaving only the previous solvent layer, which can be allowed to evaporate during centrifugation, resulting in a nearly dried surface.
  • a linearly grooved surface can be used to encourage the formation of arrays of ssDNA that are substantially parallel, substantially inline each from one end, and substantially equally spaced apart.
  • the raised portion of the grooved surface can be comprised of hydrophobic molecules which repel the highly negatively-charged master ssDNA and force their alignment onto positively-charged "gutters.”
  • a functionalized surface can be constructed by 1 ) photolithographic fabrication of gold lines in the (x) direction on a metal or polymer surface, approximately 25 nm wide and 50 nm apart, equal to just under the fully-extended length of the master ssDNA used; 2) exposure of the gold to hexadecanethiol (HDT, formula: CH 3 -(CH 2 ) I i-SH) which forms hydrophobic "risers" on the (x) direction; and 3) functionalization of the intervening troughs to have a positive charge for binding to ssDNA
  • stretching can be performed on a curved surface with radial signature relative to the length of the ssDNA strand to be stretched of between 1 and 2.5 milliradians, for example. For a 10,000 base long ssDNA strand, this corresponds to a cylinder of between 0.5-1.0 mm diameter.
  • a distal surface that falls downwards creates more opportunity for the portion of ssDNA nearer to the bead to become straightened, as opposed to a flat surface where that distal portion of the DNA has less conformational space in the (+y) direction;
  • a curved surface has fewer depressions than an imperfectly flat surface, into which depressions in the (-y) direction a portion of the ssDNA can become trapped and cease to be stretched or aligned;
  • a curved surface enables centrifugation not only in the (+x) direction, as described above, but also radially, which again takes advantage of the inertial mass of the bead to help straighten and align the ssDNA; and
  • the horizon described forms a natural pulling vector in the (+y), or upwards direction, on the ssDNA, which facilitates stretching and aligning by pulling the ssDNA away from the surface (the surface is cationic and,
  • the prepared grid can be fixed and stained using standard TEM protocols and visualized under TEMicroscopy. If properly assembled, the TEM will show straight lines of beads parallel to the side wall, indicating that substantially all of the ssDNA molecules are mounted (doormated) as desired and that they are stretched uniformly (the beads will be inline if the ssDNA strands are of equal length - however ssDNA of different lengths can also be used).
  • the distance from the line-of-beads to the gold wall will be approximately that of the theoretical length of fully stretched ssDNA - approximately 0.5 nanometers for every base unit, or about 3000 nm for a 6000 base-long ssDNA that is mounted and stretched per the above methods and variations thereof.
  • FIGS. 6A-6E show a schematic illustration of a method to prepare a template-based system from the master DNA system of the type described above, also referred to herein as Single Strand Template Manufacturing (SSTM).
  • SSTM Single Strand Template Manufacturing
  • the master DNA system comprises substantially stretched, straightened and parallel single strand DNA molecules (e.g., several thousand strands each being 6000 nucleotides in length and comprising the same sequence) which are fixed onto TEM plastic.
  • FIG. 6A shows a single strand of DNA 12 of the master DNA system 10 that can be prepared with standard nucleotides (Ae., non a-S nucleotides) after the plastic 18 carrying the affixed master DNA is removed from the gold TEM grid and mounted securely for further processing.
  • standard nucleotides Ae., non a-S nucleotides
  • Short alpha-Sulfur ssDNA oligonucleotides 26 that comprise the entire complement of the master DNA sequence are allowed to hybridize to the master DNA 12 under conditions that promote such hybridization. These oligonucleotides carry a sulfur atom in place of one of the oxygens on the phosphate group and are also referred to as alpha-Sulfur (a-S) oligonucleotides. Short a-S oligonucleotides can generally be produced by standard phosphoramidite DNA synthesis. These short oligonucleotides can be phosphorothioate-modified on their 5'-phosphate backbone.
  • the short ssDNA oligonucleotides can be 30 nucleotides (nts) in length, or 0.5% of the master sequence.
  • the length of this oligonucleotide is not limited to 30 nts and can be any suitable length.
  • the a-S oligonucleotides will bind to a gold template surface, as will be described later. However, other coupling chemistry and template surfaces can also be used.
  • a-S oligonucleotides can bind to a maleimide-coated surface or amine backbone oligonucleotides can bind to an aldehyde-coated surface.
  • biotinylated backbone oligonucleotides can bind to an SA-coated surface.
  • each oligonucleotide 26 (100,000 in total to correspond to the complete complement) can be hybridized to its complement DNA sequence on the master DNA strand 12.
  • the resulting template DNA strand 28 comprises a plurality of oligonucleotides atop a straight, continuous master DNA sequence, but addressed uniquely at each location.
  • each ssDNA molecule (at 6000 nts) of a master DNA system will hybridized to approximately 200 a-S ssDNA oligonucleotides.
  • the skilled artisan can determine the optimal concentration of ssDNA oligonucleotides to prepare a template-based system to complement the master DNA system having strands preferably being 50 nm (bead diameter) apart.
  • the solvation can then be changed to one that preserves: (i) the fixation of the master DNA strands 12 on the plastic 18, and (ii) the hybridization of the a-S oligonucleotides 28 on the master DNA strands 12, while also allowing the a-S backbone to bind to a gold surface.
  • the solvation can be changed to 50 mM Na3Citrate / 10 mM NaCI / no EDTA, pH 7.4 (the ssDNA on the dried surface being equilibrated to this buffer). As shown in FIG.
  • a cleaned gold surface 30 can then be pressed onto the side of the plastic film 18 containing the DNA 12, thereby sulfur-bonding the a-S oligonucleotides 32 to the gold 30 and also in a manner that replicates the original straight orientation of the master ssDNA 12 onto the template DNA 28 forming a template DNA system having a structure that is the mirror image of master DNA system 10.
  • the gold surface 30 can be made by a number of different techniques, most commonly vapor deposition of gold onto titanium- coated glass, polymer or another metal. Any suitable source of gold surface or method for preparing such gold surfaces is contemplated by the present invention.
  • the system can be heated to a temperature sufficient to break the hybridization bonds and release the master DNA system.
  • the gold surface 30 containing the transferred mirror-image a-S probe ssDNA 32 is referred to as the template-based DNA system 36.
  • the master DNA system can be prepared directly as a template DNA system 36.
  • the master DNA system can be prepared with a-S modified single strand DNA 34.
  • the master DNA system and the template DNA system are the same.
  • a-S ssDNA 34 can be prepared using any known or suitable method in the art, such as, for example, polymerase chain reaction (PCR) using a-S deoxyribonucleotide triphosphates (a-S dNTPs) in place of standard dNTPs.
  • PCR polymerase chain reaction
  • a-S dNTPs a-S deoxyribonucleotide triphosphates
  • 10,000 copies of 6000 nt-long a-S ssDNA can be stretched and straightened across a surface as described in the above master DNA preparation example and then bound directly to a cleaned gold surface 30 to provide a template DNA system 36 as shown in FIG. 6D.
  • the different means of solvation, surface charge density of the plastic, electric, magnetic and inertial fields can be modified for optimization of stretching and affixing a-S ssDNA as opposed to regular ssDNA. Such modifications can be determined without undue experimentation by one of ordinary skill in the art.
  • the template DNA system can be assessed for quality (e.g., tested to be sure the DNA strands are substantially parallel, straightened and stretched).
  • a partial mirror-image copy, or partial complement can be produced. This can be done by addition of 50 nt-long, 5' and 3' biotinylated probe oligonucleotides to the template and allowing them to hybridize to the template DNA array.
  • ssDNA oligonucleotides that are approximately 50 nt-long can be modified with the molecule biotin on both ends (5' and 3').
  • the length of these probe oligonucleotides is not limited to 50 nts and can be any suitable length.
  • the solvation of the system can be changed to one that preserved the hybridization of the probes to the template DNA system but facilitates binding of the probe oligonucleotides to a subsequent QA surface.
  • the solvation can be changed from 1OmM phosphate buffer / 10OmM NaCI / 2OmM EDTA, pH 7.4 to 10 mM phosphate buffer / pH 6.8.
  • the QA surface can be a thin plastic film that is transparent under TEM, and that is also positively- charged to promote binding of the negatively-charged probe oligonucleotides. This plastic can be either pressed-onto the hybridized template DNA probes or allowed to polymerize from a liquid state.
  • the template can be placed upside-down onto a droplet of Butvar in water, or the Butvar can be layered on top of the template and another TEM grid can be incorporated to hold the Butvar for subsequent QA analysis by electron microscopy.
  • the system can be heated hot enough to break the hybridization bonds, but not hot enough to alter the orientation of the probe oligonucleotides or to prevent binding of the probe oligonucleotides to the plastic.
  • the grids used for QA do not have to be gold, but can be, for example, nickel or copper.
  • the TEM plastic film containing the probe oligonucleotides can be carefully peeled-off the template and electron-dense probe elements can be added to visualize the geometrical orientation of the probe oligonucleotides that themselves mirror-image the template DNA system.
  • 10-nm diameter gold beads covered with streptavidin can be allowed to bind to the biotinylated probes on the film under conditions that promote biotin-SA bonds. The unbound excess can then be washed- off.
  • the film can be fixed, stained and the locations and orientation of the probe oligonucleotides can be determined by visualization of the SA gold beads under TEM.
  • the quality of the template DNA system, and the integrity of the process that generated the complementary copies, can determined by the location and geometric orientation of the "string of pearls" comprised of the 10 nm gold beads bound to each end of the probe oligonucleotides. Generally, a straight lines of beads in the TEM will indicate a well-manufactured template suitable for further use as described below.
  • platform DNA systems from a gold-based ssDNA template.
  • the platform DNA systems conceptually are a mirror image of the template DNA systems. These platforms can be either functionalized to perform desired tasks, or to serve as intermediates in the production of additional templates.
  • the platform systems which can be on a plastic or other robust substrate, may be more suitable for peptide manufacturing applications.
  • This example description of the platform DNA system follows the preparation of the exemplary template DNA system as described above, but uses a template DNA system that can be generated from a ssDNA library of 100 different ssDNAs, each having a unique, distinguishable, and non- redundant - however known - DNA base sequence.
  • the lengths of the ssDNA strands can also vary from about 500 to about 10,500 nucleotides in length, with a minimal size difference of about 100 bases between the different ssDNA strands of the library.
  • This library can be conveniently generated from public domain DNA plasmids and constructs or by any other suitable methods.
  • template DNA 100 different, master ssDNA strands ranging in size from 500 to 10,500 nucleotides long, can be added to modified gold TEM grids and stretched across a plastic surface as described above. A permanent template DNA system on-gold can then be generated as described above. A complete mirror image copy (100% complement) of the template, which is referred to as the "platform DNA system,” can be prepared as described below.
  • the ssDNA template system can be carefully cleaned and the solvation changed to one that promotes hybridization. 30 nt-long ssDNA oligonucleotides that comprise the entire complement of the template DNA system can then be added to the template system. After hybridization and washing, the solvation can be changed to one that preserves hybridization but which is appropriate to a high melting temperature and high-strength elastomeric material.
  • Candidate elastomers that have these properties include dimethacrylate and diacrylate resins, polycaprolactones, and surface-modified polydimethyl- and polyvinyl-siloxanes.
  • a thin (-0.5 mm) layer of the high-melt / high-strength elastomer is allowed to flow onto the template DNA system hybridized with the entire complement of ssDNA oligonucleotides, and allowed to fill-in all the gaps between and encapsulate the strands.
  • a hard backing can be bonded to it via an adhesive.
  • the hard backing can be another elastomer to serve as an even harder backing for insulation and handling of the first elastomer.
  • Candidate hard backing elastomers include polyurethanes, polystyrenes, and polypropylenes.
  • the adhesive preferably binds both hydrophilic and hydrophobic surfaces.
  • the elastomer(s) can be further constructed on a substrate, such as a polyimide, ceramic, or glass.
  • the platform can then be heated hot enough to denature the hybridization bonds between the template ssDNA and the ssDNA oligonucleotides on the solidified initial elastomer, and the platform DNA system can be carefully removed from the template.
  • a photo-polymerizable elastomer having an even smaller drop size and lower viscosity than the one described above can be used to fill-in the troughs in which the master ssDNA and hybridized compliment oligonucleotides are located.
  • a photolithographed gold layer (atop a pre-lithographed layer of, for example, titanium) can be upwards of 50 nm in height, Thus, the polymer in its liquid state should be able to flow into and reach the 30 nt oligonucleotides at the bottom of each trough.
  • the elastomer since the elastomer is charged, with individual monomers likely having a high dipole moment, the elastomer may be strongly repulsed by the 12-carbon saturated alkanes of HDT that form high density "bristles" around each raised section. This is another reason why the elastomer in this case should flow and fill small volumes with ease. Depending on the thickness of elastomer necessary to accomplish these tasks, it is also desired that the elastomer absorb all UVC light used for polymerization else stray light reaching the hybridized master ssDNA and 30 nt oligos form undesired covalent bonds or otherwise damage the complement DNA.
  • each master DNA strand is preferably separated from its neighbor by approximately the diameter of a magnetic bead, e.g. 50 nm.
  • TEMicroscopy suggested that approximately 100,000 DNA strands were on each template DNA system. Therefore, the size of a platform DNA system prepared from such a template DNA system was about 2000 nm or 2 microns wide, not counting peripheral areas, or "margins,” that lack DNA but are necessary for handling and manipulation.
  • the platform can be generated from a template that was itself generated from a master that is composed of substantially straight (e.g., R squared value of 0.99 and above) and stretched ssDNA strands.
  • each strand on the platform can be QA determined in the following manner.
  • a probe library of end-biotinylated, 50-mer oligonucleotides that are exact complements of the first and the last 50 nt of each master DNA strand and thus will bind to the first and last 50 nt of each platform strand can be hybridized to the platform, functionalized with 10-nm gold beads, and visualized using TEM as previously described. This procedure can reveal the identity of each strand via its extremities and define its length.
  • each strand can be verified by using an additional probe oligonucleotides that bind to one or more 50 nt long sequences in the intervening sequence of each strand. Therefore, the DNA base sequence of each "address" on the platform DNA array can be determined.
  • the SSTM method described above can be used to create any one- or two-dimensional structure at nanoscale levels.
  • SSTM functions analogously to the way living organisms produce proteins, and to the manner in which evolutionary pressures improve the activity and resilience of enzymes.
  • SSTM dispenses with the messenger RNA step and synthesizes enzymes and other biologically active polymers directly from DNA.
  • the template masters can be constructed from single strands of DNA which have been straightened with geometric regularity and permanently embedded upon a flat surface. Production templates or platforms, that facilitate the actual synthesis and are near perfect complementary copies of the original masters, can then be mass- produced.
  • Amino acids can be coupled to DNA units (e.g., nucleotides) to form chimaeras that can be addressed to the template or platform DNA system to synthesize or assemble a polypeptide.
  • DNA units e.g., nucleotides
  • the amino acids can be coupled to nucleotides in a manner that preserves the biological activity of both.
  • the monophosphate versions of the four natural deoxynucleotide bases of DNA can be coupled to any natural or synthetic amino acid, via use of cystamine or other linking agents.
  • FIG, 7 shows the reaction of deoxyadenosine monophosphate with arginine via a cystamine linkage to provide a chimaera of the amino acid linked to the nucleotide.
  • a library of chimaeras comprising natural amino acids can be coupled to individual DNA units in the form of deoxynucleotidyl monophosphates (dNMPs). These dNMPs are referred to herein by their bases: dAMP, dTMP, dGMP and dCMP.
  • cystamine can be coupled to the 5'-phosphate groups of all four dNMPs using standard carbodiimide linking chemistry, using the molecules EDC and imidazole. Cystamine has a disulfide bond (-S-S-) in its middle and amines (- NH 2 ) on each end.
  • TMEA trityl-mercapto-ethylaldehyde
  • Trt-S-CH 2 -CH 2 -COH molecular formula: Trt-S-CH 2 -CH 2 -COH, where Trt is a trityl protecting group residue
  • Trt-S-CH 2 -CH 2 -COH molecular formula: Trt-S-CH 2 -CH 2 -COH, where Trt is a trityl protecting group residue
  • TMEA is a coupling molecule, synthesized from available reagents, which has an aldehyde group (-CHO) on one end and a trityl-protected thiol group (-SH -> -S-T rt) on the other.
  • -CHO aldehyde group
  • -SH -> -S-T rt trityl-protected thiol group
  • each cystamine- coupled dNMP is mated to each MEA-coupled amino acid individually, resulting in a library of 76 different amino acid-dNMP molecules.
  • amino acid and nucleotide identities e.g., MET-dCTP
  • linker group composed of residual cystamine and MEA implied, though not referred-to, in the acronym.
  • monomers comprising other combinations of amino acids, nucleotides, and linker molecules (which may or may not be easily cleavable by standard methods), are herein also referred to as chimaeras.
  • chemistries can be used to bind the amino acids to the nucleotides to form the chimaeras.
  • ethylenediamine can be used to form a permanent (Ae., non-redox-cleavable) chimaera.
  • the N'-terminus of the amino acid can be coupled to the carboxyl group of bromoacetic acid (the carboxyl group of the amino acid can be protected from undesired coupling, for example, by conversion beforehand to the methyl ester) wherein the bromine atom has undergone halide displacement to one end of cystamine. This results in an amide nitrogen on the amino acid, which can't be further coupled, and a secondary amine on the linker molecule, which can be coupled.
  • the polarity of bromoacetic acid can be switched such that the N'-terminus of the amino acid has undergone halide displacement with bromoacetic acid and the carboxyl group of bromoacetic acid has been coupled to one end of cystamine. Further coupling will then occur only on the N'- terminus of the amino acid, which is now a secondary amine.
  • the nucleotide will be linked to cystamine first forming a phosphoramidite and the other end of cystamine, a primary amine group, will either be condensed directly to the N-terminus of an amino acid, undergo halide displacement with bromoacetic acid, or be coupled to bromoacetic acid and the amino acid will be linked. Since any primary or secondary amine group, electron rich nucleophile, or carboxylic acid, is susceptible to premature coupling, the relevant amino acids as well as the nucleotides having such groups can have those groups protected using orthogonal protection and deprotection schemes familiar to those practiced in the art of Solid Phase Peptide Synthesis (SPPS).
  • SPPS Solid Phase Peptide Synthesis
  • HATU-mediated coupling of freely jointed secondary amines (which does not include Proline, a secondary amine stiffened as a result of cyclization), is slowed by the ability of constantly-in- motion N-acyl side groups to inhibit covalent binding to activated C-termini by Van der Waals and other factors.
  • the resulting amino acid-nucleotide chimaeras are then addressed to the single strand DNA template in a manner consistent with Watson-Crick base pairing rules.
  • the amino acids can then be peptide-bonded to form either small peptides or to serve as the subunits of larger protein-based molecules.
  • the DNA nucleotide residues are preserved on the polymerized amino acid subunits, facilitating their addressability to other templates in a subunit sequence specific manner.
  • the chimaeric molecules can be chemically treated to uncouple the polypeptide portion from their nucleotide carriers, leaving chains of amino-acids that, upon purification and qualification, are protein-based mimetic enzymes.
  • FIG. 8A shows the basic concept of bonding of a chimaera 46 comprising an amino acid 44 and the DNA unit 42 to a template DNA strand 32 by nucleotide pairing.
  • FIG. 8B shows a fully realized polypeptide sequence 48 as attached to the template DNA strand 32.
  • One permanent single strand DNA master can exponentially generate a large number of single strand DNA production templates, which can then act as the foundation for synthesis of a wide variety of polymer molecules.
  • a particular spatial orientation can be forced on a polypeptide-based product in order to solve the folding problem prevalent within the construction of synthetic enzymes.
  • artificially manufactured polypeptides are limited in both their size and usefulness because of the lack of ability in the current art to elicit conformational shapes in peptides.
  • a casual review of the literature and of manufacturers' catalogs reveal few if any peptides larger than 50 amino acid residues in length that guarantee biochemical and/or enzymatic activity.
  • SPPS is more than able to produce polypeptides in excess of 50 residues in size, the ability to fold such polypeptides properly into active molecules remains problematic.
  • the three-dimensional structure of a given protein-based polymer can be controlled by selectively including or excluding the participation of a nucleotide and the molecules which serve as linking agents to the amino acid, i.e., cystamine and its derivatives.
  • Standard amino acids can be used as monomers in synthesis if a three-dimensional structure prediction or determination has indicated it would be best to use them for protein folding (orthogonal protection, if necessary, is reasonably implied).
  • bond-forming elements within the linking agents that bind to any combination of: (i) other linking agents, (ii) the reactive side chains of amino acid residues, and/or (iii) to a pre-formed solid surface or liquid-liquid interface, can be used to solve the folding problem.
  • Residual molecules formerly linking amino acids to nucleotides can help manage the folding of polypeptide-based polymers into desired conformations, using molecular structures centered on the secondary amine "tails" that are artificially generated upon cleavage of the polypeptide-based product from its carrier nucleotides. For example, once the reduction-cleavable disulfide bond in the cystamine residue is severed, a polypeptide product is left with one or more secondary amine groups, that are distinct from the "natural backbone" N'-to-C of the product and that terminate in thiols, or mercaptyl, groups.
  • modifications to the standard amino acid monomers can provide polypeptides that have a large number of covalent links that form a secondary backbone, or "biomimetic skeleton," and products having such a structure are heretofore referred to as mimetics.
  • This additional structure is formed by the condensation of proximal thiol groups into disulfide bonds under conditions familiar to those skilled in the art, e.g., under oxidizing conditions.
  • This additional structure on the polypeptide-based polymer can add resilience in excess of naturally-produced proteins having the same amino acid sequence.
  • enzymes and other polypeptide-based molecules to resist extremes of temperature, pressure, pH, salt and shear forces which characterize industrial processes and otherwise degrade the enzymes.
  • Enzyme mimetics with preserved catalytic activity under trans-biotic conditions that would neutralize most naturally-occurring enzymes can be achieved.
  • "Active Site Only" enzyme-like mimetics can be synthesized that do away with the mostly non-catalytic portion of the polypeptide and replace that with a stronger organic or inorganic scaffold.
  • the enzymes can be scaffolded, in whole or in part, to artificial surfaces. Therefore, functionalization of solid surfaces that facilitate enzyme-based industrial processes and biomedical components with long lasting catalytic protein mimetics can be achieved.
  • FIGS. 9A and 9B show an exemplary method (Steps 1-12) of synthesizing a polypeptide using a template DNA system, wherein the resultant polypeptide is covalently linked to its template DNA strand and can be a free polypeptide or can be complexed to a secondary skeleton via a scaffold with DNA binding capacity.
  • Step 1 shows a template DNA 52 bound to a gold surface 54 via (P)-thioate bonds (indicated by asterisks * ).
  • the template DNA 52 can be 5'-dephosphorylated to enable easier bonding to gold.
  • the template DNA 52 in this example comprises a short 28 nt long a-S ssDNA sequence of unique sequence. The last 16 nt (counting from the 5'-to'3' direction) will serve as the literal "template” from which amino acids will be addressed, and polypeptides produced.
  • the gold surface 54 can comprise any suitable gold surface, such as the flat gold surface described previously used to make a template DNA system.
  • the surface 54 can comprise gold beads of approximately 30 nm in diameter.
  • Such gold beads can be formed by prior art reduction of gold chloride (HAuCI 4 -3H 2 0) in citrate buffer. The resulting "colloidal gold” can then be precipitated with ethanol and resolvated to facilitate the binding of a template DNA. The gold beads can then be washed and standard testing (spectrophotometry that measures the amount of single strand DNA, and other methods) can then be performed to verify that the template DNA is on the gold beads.
  • the persistence length of ssDNA in citrate buffer is such that the a-S oligonucleotides will bind straight and flat enough, backbone side down, for anchoring and templated synthesis to occur on the gold bead surface.
  • bead surfaces such as maleimide-coated and lithographically structured surfaces can also be used.
  • Step 2 shows a modified anchor DNA 56 added and allowed to hybridize to the template DNA 52.
  • the 12 nt long ssDNA anchor sequence is complementary to the first 12 nt of the template DNA and is modified on one end to accept a first amino acid.
  • the 5' end of the anchor DNA is functionalized with cystamine to present a free amine (-NH 2 ). This results in a cleavable disulfide bond and that coupled to the 5'-end of the anchor DNA using carbodiimide chemistry.
  • the resulting anchor has a free amine on its 5'-end that is also cleavable under reducing conditions to decouple the amino acids from the anchor.
  • Step 3 after washing and determination that the anchor is properly on the template (e.g., via spectrophotometry that measures the amount of double strand DNA, and other methods), the solvation is changed to an environment preferential to the creation of inter-strand cross-links (indicated by W) to covalently attach the anchor to the template.
  • the molecule psoralen can be added to the gold beads with the template DNA and anchor DNA. Psoralen can form permanent covalent bonds between the anchor and template ssDNAs, upon proper solvation and dosing with UVA light (365 nm wavelength). After the solution is UVA-exposed, the psoralen is washed away.
  • the 16 nt-long "template" sequence in this example is comprised of four repetitions of the sequence G-C-A-T in the 5'-to-3' direction. Amino acids are thus addressed to this unit sequence in the repeating order 3'-C-G-T-A-5'.
  • Polypeptides of any reasonable length e.g., from 64 to nearly 10,000 amino acids long
  • Longer polypeptides can be created using DNA templates not on beads, but on a template-based system on a flat surface, as described above, which better facilitates the addressing of such subunits on more geometrically arrayed, i.e., straighter, ssDNA template strands.
  • polypeptides can be synthesized according to the following Steps 4 to 12.
  • Step 4 shows the base-specific 5'-to-3' addressing and amidation of the first coupled amino acid-nucleotide chimaera 58 (e.g., MET-dCMP) to the first template address (G1 ).
  • MET-dCMP first coupled amino acid-nucleotide chimaera 58
  • This example uses ethylenediamine-coupled chimaeras.
  • the coupled amino acid MET-dCMP can have its C'-terminus activated with the molecule HATU under solvation conditions that promote such activation, using familiar SPPS methods in which the monomer is pre-activated and added to the template.
  • the MET- dCMP presents a protected N'-amine to prevent self- polymerization as shown by the encircled (-NH 2 ) group.
  • the solvation of the template can then be changed to one that is both (i) compatible to the HATU-activation, and that also (ii) promotes hybridization of DNA bases.
  • the activated MET-dCMP can then be added to the template beads and formation of a peptide bond between the MET-dCMP and the free amine on the 5'-end of the anchor sequence occurs readily.
  • the excess monomeric chimaera can be washed-off and the template re- solvated to conditions compatible with deprotection.
  • Step 5 shows subsequent addressing and amidation of the second to the fourth amino acids (C to N'): Arg, Ser, and Tyr, to the complements of their dNMP couples on the template (5' to 3') having locations: C1 , A1 , and T1.
  • the second DNA-amino acid e.g., ARG-dGMP
  • ARG-dGMP can be C- activated with HATU as above, added to the template as above, washed and its N-terminus deprotected from Fmoc.
  • the third and fourth DNA-coupled amino acids e.g., SER-dTMP and TYR- dAMP, can be added to the template and treated similarly.
  • This four amino acid long polypeptide, still coupled to its DNA carriers (composed of four unique DNA bases) is referred to as a 'packet' 60, the smallest size polypeptide-based polymer of significance, and is a non-autonomous part of a subunit.
  • the template sequence be comprised of tetranucleotide repeats, as shown, in order to maximize the distance between similar bases (Ae., all bases are separated by three dissimilar bases from themselves). This minimizes the chances of an addressed chimaeric molecule being coupled when not base-paired to the base directly 5'-adjacent to the growing polymer, e.g., MET- dCMP having first been coupled while addressed to location (G2), and not location (G1 ) as desired.
  • tetradon a repeating tetranucleotide comprising a portion of the template, and which can comprise any single occurring combination of the bases A, T, G and C.
  • the first tetradon is 5'-G-C-A-T-3'. This term is chosen for comparison and distinction with "codon,” the commonly used term for a trinucleotide sequence.
  • codon the commonly used term for a trinucleotide sequence.
  • a given codon will only code for one amino acid.
  • a given tetradon sequence can code for (considering natural primary amine amino acids only, of which there are nineteen) 19 to the fourth power different amino acid sequences of packets, equal to 130,321 different combinations.
  • Step 6 further addressing and amidation of the 5 th to 16 th amino acids (unspecified, designated N) forms an initial 16-amino acid-long subunit 62, referred to as Subunit N.
  • the subunit is delinked from the anchor at its C-terminus by reduction, presenting a free thiol group, and dehybridized from the template 52.
  • the dehybridized subunit 64 is now autonomously addressable. In the example shown here, the couplings to dNMPs are not redox- cleavable.
  • the covalent nature of the anchor-to-template bonds, and that of the first amino acid (Methionine) to the 5'-end of the anchor helps to keep the nascent polypeptide chain on the template and helps to promote hybridization of the DNA portion of the amino acid chimaeras onto the template DNA.
  • the MET portion of the resulting polypeptide subunit 64 is complexed with an uncleavable cystamine residue on its C- terminus, i.e., a mercaptoethyl group (HS-CH 2 -CH 2 -N'-terminus of MET residue) even after reduction to cleave the subunit from the anchor.
  • This amidated residue will prevent further peptide bonding and the creation of longer polypeptides.
  • the first amino acid can be removed by treatment with Carboxypeptidase B.
  • the first amino acid can be removed by treatment with Carboxypeptidase A.
  • Step 9 shows the 15-mer subunit deletion fragment 66 after treatment with a carboxypeptidase to remove the first amino acid addressed to the subunit (Met) and present a free C'-terminus carboxyl group on the second amino acid (Arg).
  • Step 10 shows the 16- ⁇ mer subunit 68 after HATU activation of the C'-terminus of Step 9 and re- amidation with Met.
  • another amino acid can be used, and with another dNMP couple. In this example, both the original residue and the original nucleotide couple were preserved.
  • the intact subunit 68 is addressed to a longer template 70 (e.g., a gold foundation and a-S, or phosphorothioate bonds implied as in Step 1 ), via base-pair specific bonds.
  • this subunit represents "Part N" of a larger polypeptide that can be made up of an "alphabet" amount of subunits addressed on the template either behind (C, implying a Subunit M not shown in the figure), or in front (N', representing Subunits O, P, etc.) to this subunit.
  • Step 12 shows subsequent addressing and amidation of the 0 th and P th subunits in the C'-to-N' direction on the template, to each other and to Subunit N.
  • All subunits can be preactivated with HATU and added to one at a time to a pre-addressed subunit on the template that has been N-terminus deprotected.
  • This pre-addressed subunit can optionally be "Subunit A" and still be disulfide bound to the anchor sequence, which promotes more efficient production due to the covalent bond of Subunit A to the anchor, which cannot be broken under the different solvation conditions, and variations thereof, as described above.
  • the template DNA sequence comprises "quartets of tetradons," i.e., subunits of 16-mer sequences comprising four repeats of the same tetradon. This design facilitates the correct addressing and orientation of each polypeptide-based polymer subunit to the template.
  • the N'-terminus can be deprotected of Fmoc and the product complexed to a secondary skeleton via, in this example, a scaffold with DNA binding capability.
  • a scaffold with DNA binding capability will comprise a two- or three-dimensional surface derivatized with nucleotides and/or bases that have been pre-formed into geometrical patterns. The patterns of such A,T,G, and C on the solid surface will facilitate the final three dimensional conformation, via folding, of the polypeptide product.
  • FIGS. 10A, FIG. 10B, and FIG. 10C together show another exemplary method (Steps 1-15) of synthesizing a polypeptide using a template DNA system, wherein the resultant polypeptide is covalently linked to its chimaeric nucleotide groups through cystamine linkages, which can be utilized to complex to a secondary backbone for scaffolding purposes.
  • Step 1 shows a 28-mer a-S ssDNA template 52 bound to a gold surface 54 (indicated by the thick line) via alpha-S, or phosphorothioate bonds (indicated by the asterisks * ), as previously described.
  • Step 2 shows a 12-mer anchor 56 hybridized to the template 52.
  • the anchor 56 is 5'- functionalized with ethylenediamine (H 2 N-CH 2 -CH 2 -NH 2 ) phosphoramidated to the phosphate group of the 5'-Deoxyguanosine of the anchor) presenting a free amine (-NH 2 ).
  • Step 3 shows inter-strand cross-link covalent bonding of the anchor 56 to the template 52 via UV radiation.
  • Step 4 shows base-specific 5'-to-3' addressing and amidation of the first amino acid Methionine, coupled via cystamine to its nucleotide carrier, forming the chimaeric molecule 72 (MET- dCMP), base-paired to the first template address (G1 ).
  • (Met) was pre-activated on its C'-termini with HATU and presents a protected N'-amine.
  • Step 5 shows subsequent addressing and amidation of the second to the fourth amino acids (C'-to-N'): Agr, Ser, and Tyr, to the complements of their dNMP couples on the template (5'-to-3'): C1 , A1 , and T1. This results in a non-autonomous packet 74 of four amino acid residues.
  • Step 6 shows further addressing and amidation of the 5 th to 16 th amino acids (unspecified) to form a 16-amino acid long subunit 76.
  • the subunit is delinked from the anchor at its C via a DNA-depolymerizing nuclease, of which P1 Nuclease is an example, presenting the (G1 ) nucleotide still animated to (Met) and dehybridized from the template.
  • the dehybridized subunit 78 is now autonomously addressable.
  • the couplings to dNMPs are cleavable under highly reducing conditions.
  • Step 9 shows the 15-mer subunit deletion fragment 80 after treatment with a carboxypeptidase to remove the first amino acid addressed in the subunit (Met) and present a free C'-terminus carboxyl group on the second amino residue (Arg).
  • Step 10 shows the 16-mer subunit 82 after HATU activation of the C'-terminus of Step 9 and re- amidation with Met.
  • another amino acid can be used, and with another dNMP couple.
  • both the original residue and the original nucleotide couple were preserved.
  • Step 11 shows addressing of the intact subunit 84 to a longer template 86 (e.g., a gold foundation and (P)-thioate bonds implied as in Step 1 ), via base-pair specific bonds.
  • this subunit represents "Part N" of a larger polypeptide made up of an "alphabet" amount of subunits comprised of subunits both C to (not shown) and N' to (Subunits O and P) this subunit.
  • the template comprises quartets of tetradons: 5'-(GCAT) 4 - (AGTC) 4 -(TACG) 4 -3', wherein each 16-mer subunit is assigned to a unique 16-mer quartet, via its nucleotide carriers, in order to correctly address each subunit to its correct location on the template, and also in the correct orientation.
  • Step 12 shows subsequent addressing and amidation of the 0 th and P th subunits in the C'-to-N' direction on the template, and to Subunit N. All subunits can be pre-activated with HATU and added one at a time to a pre-addressed subunit on the template that has been N'-terminus deprotected. This deprotected subunit can optionally be "Subunit A" and still be ethylene diamine bound to the anchor sequence with the manufacturing benefits of such anchoring as previously described.
  • Step 13 shows a dehybridization from the template and a decoupling from its nucleotide carriers of the polypeptide product comprising Subunits N-O-P.
  • Subsequent oxidation of the residual thiol tails that have been secondary-am inated to the N'-terminus of each amino acid results in the formation of disulfide bonds at random positions - on the average, one every two residues.
  • a free thiol group remains from the formation of a disulfide bond between every three successive pairs of residues, i.e., a free (-SH) remains at approximately every 7 th former N-terminus.
  • disulfide bond formation is largely random and driven by spatial proximity promoted by folding due to the side chains of the amino acids and the solvation conditions chosen that determine both folding as well as the kinetics of disulfide bond formation.
  • the exemplary product is simplified to a linear conformation (Ae., no folds) and one-in-seven free thiol groups, as shown.
  • Step 14 shows representative options in formation of secondary backbones formed by disulfide bonds, as well as functionalization of the free thiol groups for "scaffolding" purposes.
  • Extreme left example shows a maleimide group that has not yet bound to the free thiol in its proximity (shown for demonstration only).
  • Second from left shows a maleimide-thiol bond.
  • Middle two show direct binding of thiol groups to the gold portion of the nanobar.
  • Right two show cyclization of the last two thiol groups on the N'-terminus of the polypeptide. The sum total of all such secondary functionalizations, to a secondary scaffold or not, determine the overall 3D conformation of the polypeptide.
  • Step 15 shows further options for backbone scaffolding.
  • Subunit N residue shows base pair- specific addressing of the polypeptide to nucleobase moieties on the nanobar (UV-induced covalent bonds shown).
  • Subunit P residue shows direct gold-to-thiol bonds.
  • Subunit O residue shows maleimide-thiol bonds.
  • the shape of the secondary scaffold, base sequence and types of secondary amine tails affect the kinetics and direction of folding and, thus, determine the overall 3D shape.
  • a variation on the aforementioned exemplary method of synthesizing polypeptide-based polymers from an ssDNA template involves the activation of carboxyl groups on the solid phase and the subsequent addressing of amino acid-nucleotide chimaeric monomers such that polymerization is achieved.
  • This C-terminal activation can be accomplished by the following methods:
  • Method A Activation of carboxyl groups (-COOH) and/or C-termini of amino acid residues in the solid phase by hydroxybenzotriazole-based activation agents (e.g., HATU) under organic solvation conditions, e.g., DMF, NMP, DMSO.
  • hydroxybenzotriazole-based activation agents e.g., HATU
  • organic solvation conditions e.g., DMF, NMP, DMSO.
  • Method B Activation of solid phase (-COOH) carboxyls by carbodiimide- or morpholinium-based activation agents (e.g., EDC, DMT-MM) under aqueous or alcohol solvation conditions, e.g., amine-free phosphate buffers like MES, or methanol.
  • carbodiimide- or morpholinium-based activation agents e.g., EDC, DMT-MM
  • aqueous or alcohol solvation conditions e.g., amine-free phosphate buffers like MES, or methanol.
  • Method C Conversion of solid phase (-COOH) to acid chlorides (-COCI) by the use of thionyl chloride or phosphoryl chloride, under strictly organic solvation conditions, e.g., DCM.
  • the general strategy for this involves the generation of a Vilsmeir-Haack intermediate via catalytic amounts of DMF, and also stabilization of the reaction through the presence of tertiary amines such as DIPEA and piperidine.
  • Method D Conversion of the solid phase (-COOH) to anhydrides (-CO-O-OC-Ac) by the use of Acetyl Chloride under strictly organic solvation conditions, e.g., DCM, CCI4.
  • carboxyl anhydride-based coupling is the stoichiometric loss of one half of the amine-based monomer to displacement of the Acyl-acid group at each coupling step.
  • the main advantage of performing N-to-C direction synthesis on the ssDNA template system is that the need for N-terminal protection and deprotection is eliminated.
  • Monomers of amino acids (all naturally occurring ones including Proline, and unnatural ones), similarly chimaeric molecules having secondary amine groups, or other molecules having amine groups (and acid groups if further polymerization is desired), can iteratively bind to activated ester carboxylic acids, acid halides or acid anhydrides on the template.
  • Another advantage of performing N-to-C direction synthesis is that activation of the solid phase, in contrast to activation of (-COOH) in the liquid phase, eliminates the possibility of C-terminal-activated monomers coupling to cyclic or exocyclic amines on the Adenosine, Guanosine and Cytidine moieties on the template (Thymine bases lack amines entirely), or self-polymerizing to themselves via their nucleotide carriers.
  • the amines on nucleobases must be left unprotected so as to facilitate base pairing and addressing of monomers and subunits to ssDNA template systems.
  • the saturated alkane groups residual of cystamine and ethylenediamine help to support a hydrophobic interface (heretofore referred-to as the "mezzanine") directly above the vicinal water-based solvent layer that normally saturates double-stranded DNA.
  • mezzanine hydrophobic interface
  • Debye layer of hydrogen-bonded nucleobases (A to T, C to G), water molecules, salts and other ions lies the deoxyribose groups of the carrier nucleotides linked to amino acids.
  • sugar groups are less hydrophilic than the bases, yet are not strictly hydrophobic (the sugar moieties of the template DNA do not contribute to this effect as they are not only phosphate-bonded 5'-to-3'-hydroxyl, but are also alpha- Sulfur bound to the gold surface).
  • an ordered linear grouping of the chimaeric sugars forms, from bottom-to-top, a hydrophilic-to-hydrophobic mezzanine that separates the strictly "watery” layer below from, potentially, a strictly "organic" bulk phase above.
  • the aqueous layer that enables base pairings is protected from disturbance by the mezzanine, and by Van der Waals repulsion of organic solvents in the bulk phase away from the increasingly charged species (as one goes downwards).
  • coupling reactions that include solvations in NMP, methanol and DCM, as described above can still take place on a ssDNA template system without undue harm to the hydrogen bonds linking A to T and G to C.
  • coupling reactions that take place under aqueous conditions, and that do not either dehydrate or deionize the aqueous layer are preferred.
  • the couplings to the DNAs can be broken by reducing conditions (e.g., with DIT or mercaptans), resulting in a chain of polypeptides that have residual cystamine groups secondarily-aminated to each amino acid residue. This would be the case if the amino acids in question were coupled to their nucleotide carriers with the molecules cystamine and 2-mercapto-ethylaldehyde.
  • Variations of the above cases i.e., some amino acids can be linked by cystamine plus 2- mercapto-ethylaldehyde, and some by Compound 6b+2, can be used with the intention of managing the ultimate conformation, shape, stiffness, skeletal-functionalization, and other aspects of the polypeptide product.
  • a "linked chain" where each link is composed of two amino acids can be constructed by oxidizing each two successive cystamine residues (denoted by C) on a polypeptide chain.
  • (a-f) are the linkage points on a second skeleton (e.g., can be a thiol or maleimide group, or gold atom) b.
  • a stiff '"second backbone" comprising sequential disulfide bonds, can be constructed by oxidation of each cystamine residue onto a conformationally-stiff alkene chain (skeleton), which has itself been modified to carry cystamine residues.
  • a conformation whence the middle of the polypeptide sequence has flexibility (wide range of motion and conformational options), and the extremities have a stiffened character (small range of motion and fewer conformational options), can be constructed by coupling of the middle amino acids with Compound 6b+2, and the amino acids on the extremities with cystamine plus 2-mercapto-ethylamine (with reduction), followed by oxidation to form disulfide bonds.
  • linker molecules As explained above, other types of linker molecules, with additional chemistries, can be used as linkers.
  • Use of bromoacetic acid as an intermediate linker between the N-terminus of an amino acid (other than proline or its derivatives) adds further functionality and conformational options to the end- product.
  • the amino acid can be N-acylated via halide displacement, forming a carboxyl-terminated secondary amine "tail" that can be amidated to either (i-a) cystamine or (i-b) ethylenediamine residues for the synthesis of chimaeras, to (ii) amine groups on amino acid residues (Lysine, Arginine, Histidine) to promote folding/conformation, and/or (iii) amine groups on solid surfaces; (2) the amino acid can be coupled to the carboxyl group of bromoacetic acid, forming a bromine-terminated secondary amide "tail" that can be further bonded to amines as just described.
  • option (2) just described enables coupling, not on the natural backbone of the chimaeric molecule, but on the secondary backbone of the mimetic tail. Specifically, amidation of bromoacetic acid to the N-terminus of an amino acid has eliminated that location as a potential coupling point.
  • a secondary amine upon halide displacement of the bromine atom and condensation to a free primary amine (such as that from cystamine or ethylenediamine), a secondary amine has been formed on the mimetic tail which, upon exposure to a HATU-activated carboxyl group on another monomer or polymer, will form a secondary backbone that is fused, at that location, via a peptide bond and not a disulfide bond as previously described.
  • halo acids can be used as linker components, and that include other halides, e.g., fluorine and iodine.
  • the lengths of the halo acid backbones can range from ethanes to decanes, and can also be, in whole or in part, unsaturated, cyclic, and/or be further functionalized with chemically reactive groups.
  • Additional other chemistries are provided by commercially available molecules that, individually or in combinations, can serve thusly as: (1 ) linkers between amino acids and nucleotides, (2) portions of a secondary mimetic skeleton, and/or (3) braces for the attachment of the polypeptide-based polymer to a solid surface, i.e., a carapace.
  • Examples of such chemistries can be hydrazides, keto- enol enabling groups, Sn2 reactions, imine formation and condensation of diols.
  • Available reagents enable the ability to utilize, for example: (i) linkers with saturated or unsaturated linear, branched and cyclic hydrocarbons, (ii) linkers with carbonyl groups (as with the use of bromoacetic acid in one case above), (iii) linkers with primary or secondary amine groups (as with the use of bromoacetic acid in another case above), and (iv) linkers with any combinations of keto, enol, aldehyde, epoxide, carbamate, and other groups.
  • linkers that use the side groups of amino acids for inspiration can include hydrophobic groups (as in the side chains of ALA, ILE, LEU and VAL), hydrophobic groups that "stack" (as in the side groups of PHE and TYR), positively charged groups (as with ARG, LYS and HIS) and negatively charged groups (as with GLU and ASP).
  • Such linkers can have actual amino acid-like side chains as functional groups, to promote product folding by, for example as inferred above, forming hydrophobic cores, stacked ring groups, salt bridges between acids and amines, and repelled conformations between similarly-charged groups, to either other linkers or the side chains of amino acid residues.
  • linker molecules initially served as couples to link amino acids to dNMPS for addressing to an ssDNA template - for faster and more efficient polypeptide synthesis. Afterwards, they serve as the basis for creating newer and better enzymes to catalyze a wide variety of chemical processes under conditions where naturally-synthesized enzymes, or even those manufactured using standard SPPS, would degrade and become unusable.

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Abstract

L'invention concerne un système à base de gabarit permettant l'assemblage de macromolécules et de nanostructures. Le système de gabarit comprend une pluralité de molécules d'ADN simple brin qui sont sensiblement parallèles, sensiblement en ligne à partir d'une extrémité, et sensiblement espacées de façon égale, chaque molécule d'ADN ayant une longueur distinguable et une séquence connue. Le système peut être utilisé pour la synthèse précise, exacte et rentable de peptides, protéines et enzymes.
PCT/US2008/057013 2007-03-15 2008-03-14 Procédé et système pour l'assemblage de macromolécules et de nanostructures WO2008112980A2 (fr)

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EP2315822A1 (fr) * 2008-08-06 2011-05-04 Incitor Incorporated Création d'ensembles adressables multidimensionnels haute densité
US8993714B2 (en) 2007-10-26 2015-03-31 Imiplex Llc Streptavidin macromolecular adaptor and complexes thereof
US9102526B2 (en) 2008-08-12 2015-08-11 Imiplex Llc Node polypeptides for nanostructure assembly
US9354189B2 (en) 2008-12-02 2016-05-31 President And Fellows Of Harvard College Apparatus for measurement of spinning forces relating to molecules
WO2017222711A1 (fr) * 2016-06-22 2017-12-28 The Charles Stark Draper Laboratory, Inc. Composition destinée à être utilisée dans la synthèse des chaînes moléculaires
US9869851B2 (en) 2008-12-02 2018-01-16 The Research Foundation For The State University Of New York Centrifuge force microscope modules and systems for use in a bucket of a centrifuge
CN111198175A (zh) * 2018-11-19 2020-05-26 中国科学院宁波材料技术与工程研究所 热点可控分布的宏观大面积纳米金棒二维阵列及应用
US10948401B2 (en) 2016-02-25 2021-03-16 President And Fellows Of Harvard College Spinning apparatus for measurement of characteristics relating to molecules

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US8993714B2 (en) 2007-10-26 2015-03-31 Imiplex Llc Streptavidin macromolecular adaptor and complexes thereof
EP2315822A1 (fr) * 2008-08-06 2011-05-04 Incitor Incorporated Création d'ensembles adressables multidimensionnels haute densité
EP2315822A4 (fr) * 2008-08-06 2013-05-01 Incitor Inc Création d'ensembles adressables multidimensionnels haute densité
US9102526B2 (en) 2008-08-12 2015-08-11 Imiplex Llc Node polypeptides for nanostructure assembly
US9354189B2 (en) 2008-12-02 2016-05-31 President And Fellows Of Harvard College Apparatus for measurement of spinning forces relating to molecules
US9869851B2 (en) 2008-12-02 2018-01-16 The Research Foundation For The State University Of New York Centrifuge force microscope modules and systems for use in a bucket of a centrifuge
US8795143B2 (en) 2008-12-02 2014-08-05 President And Fellows Of Harvard College Spinning force apparatus
WO2010065477A3 (fr) * 2008-12-02 2010-10-07 President And Fellows Of Harvard College Appareil à force de rotation
WO2010065477A2 (fr) * 2008-12-02 2010-06-10 President And Fellows Of Harvard College Appareil à force de rotation
US10265705B2 (en) 2008-12-02 2019-04-23 President And Fellows Of Harvard College Apparatus for measurement of spinning forces relating to molecules
US8491454B2 (en) 2008-12-02 2013-07-23 President And Fellows Of Harvard College Spinning force apparatus
WO2010132363A1 (fr) * 2009-05-11 2010-11-18 Imiplex Llc Procédé de fabrication d'une nanostructure protéique
US9285363B2 (en) 2009-05-11 2016-03-15 Imiplex Llc Method of protein nanostructure fabrication
US10948401B2 (en) 2016-02-25 2021-03-16 President And Fellows Of Harvard College Spinning apparatus for measurement of characteristics relating to molecules
US11913872B2 (en) 2016-02-25 2024-02-27 President And Fellows Of Harvard College Spinning apparatus for measurement of characteristics relating to molecules
WO2017222711A1 (fr) * 2016-06-22 2017-12-28 The Charles Stark Draper Laboratory, Inc. Composition destinée à être utilisée dans la synthèse des chaînes moléculaires
US10975407B2 (en) 2016-06-22 2021-04-13 The Charles Stark Draper Laboratory, Inc. Composition for use in molecular chain synthesis
CN111198175A (zh) * 2018-11-19 2020-05-26 中国科学院宁波材料技术与工程研究所 热点可控分布的宏观大面积纳米金棒二维阵列及应用
CN111198175B (zh) * 2018-11-19 2022-09-27 中国科学院宁波材料技术与工程研究所 热点可控分布的宏观大面积纳米金棒二维阵列及应用

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